Guidance on the Inactivation or Removal of Select Agents and Toxins for Future Use: Procedure Validation

Procedure validation can be accomplished by viability, infectivity, or toxicity testing. An entity must validate their inactivation or select agent removal procedure on-site to determine that the procedure works as intended, including the use of appropriate positive, negative, and process controls. This can be accomplished by using the final inactivation conditions derived from the procedure development step (or from an existing procedure (commonly accepted or published procedure)), and testing for the absence of viable organism, infectivity of regulated nucleic acids, and/or toxicity of regulated toxins. Perform sufficient experimental replicates to determine inherent variability with the procedure. The number of replicates will vary depending on the procedure and agent, and it is the entity’s responsibility to determine a sufficient number of replicates. If practical, multiple types of assays for determining viability are recommended.

Once non-viability or non-infectivity have been demonstrated, and the record requirements are met, the material is no longer subject to the regulations. It can be moved to lower containment, transferred without approval from FSAP, used in un-registered space, etc.

Examples of items to consider when validating an inactivation or select agent removal procedure are listed below:

• Adherence to standardized extraction kit instructions (manufacturer or in- house derived) for nucleic acids, proteins, polysaccharides, etc.
• Manufacturer performance data for filter used to remove/exclude viable organism.
  • Appropriate pore size of the filter depending on organism size and matrix material (e.g. serum, culture media).
  • Volume of starting material (should not use more volume than manufacturer recommends)
• Appropriate assay for the starting material (virus, vegetative bacteria, or spores).
• LOD/LOQ of the viability testing protocol.
• Concentration of starting material containing select agent and regulated nucleic acids (start with highest concentration expected as a worst case scenario and then set that concentration as the upper limit for subsequent inactivation).
• Matrix materials that could interfere with viability tests.
• Validation with all (100%) of the sample (for large volume cultures you can filter 100% of the sample and then culture the filter)
• Need for neutralization of chemical or antimicrobial treatments.

Viability Testing of Inactivation or Removal Procedures

Validation of inactivation or removal procedures will differ depending on the category of the sample. There are three categories of sample: 1) inactivated agent, 2) extracts, or 3) material (within this section, material refers to sample in which intact select agent has been removed).

Inactivated Organism

To verify an agent as non-viable, viability testing procedures may include, but are not limited to, cell viability assays, growth analysis, in vivo exposure, or all of the above. Consider the type of viability test, like broth culture versus agar plates for bacteria. In some instances, extended broth cultures allow for the growth of sub-lethally damaged organisms (REF 1). If any failures in viability testing or inconclusive results occur, reassess procedures. Consider redeveloping the inactivation procedure if you can’t identify a variation from the prescribed procedures to explain the failure of viability testing. Inactivation failures when validated procedures are accurately followed is an indication that the procedure is not sufficiently reliable to be used for inactivation.

The LOD/LOQ of the viability testing procedures (related to the detection assay and the sampling of inactivated material) precludes demonstrating full sterility of inactivated material. Consider these limitations when 1) generating validation data, and 2) determining the extent of sampling required for subsequent inactivation (see below).

Extracts

Under ideal conditions, the presence of viable organisms in an extract (for example, nucleic acids, proteins, or polysaccharides) would be a rare event. Treatment conditions are considered extreme due to both destruction of the select agent (like lysis of select agent) and then additional steps (like filtration, precipitation, centrifugation, column capture) to further purify the nucleic acids, proteins, polysaccharides, removing any remaining viable select agents. Because these procedures require the detection of a rare event and require prohibitive sample sizes, statistical methods cannot be practically applied.

For select agent nucleic acid extracts, procedure development via kill curves is not typically performed. This also applies to other types of extracts. Instead, the entity can use a standardized nucleic acid extraction kit (from a manufacturer or derived in-house) for inactivation. Agent concentration, volume, mixing time, inactivation/extraction time, and temperature should be held constant to be considered standardized. Non-viability should be confirmed by initial in vitro viability testing using all (100%) of the sample in broth and/or agar for bacteria, plaque or cytopathic effect detection on a permissive cell line for viruses, or other appropriate culture method (e.g. chicken embryo for HPAIV) with suitable positive and negative controls.

If after using the standardized nucleic acid extraction kit, viability is still detected, conduct a separate filtration step using the appropriate pore size filter. Test the filtrate using all of the sample to confirm non-viability during validation. If non-viability is confirmed then this additional step should be added to the inactivation procedure. This should satisfy inactivation and viability testing validation requirements without the need for establishing kill curves. If viability is still detected with the use of a standardized nucleic acid extraction kit and the additional filtration, this procedure should not be used for inactivation.

Removal of viable agent

Within this section, material refers to sample in which intact select agent has been removed. This is not to be confused with extracts which refers to nucleic acids, antigens, lysates, or select agents that have been subjected to lysis before removal. Using a standardized filter with the appropriate pore size for removing a select agent from material, validation by viability testing all (100%) of the sample with an appropriate culture method and positive and negative controls, and verification viability testing on a portion of subsequent samples (see verification section for more information) should satisfy the validation requirements without the need for establishing kill curves.

Infectivity Testing of Inactivation Procedures

An infectivity testing procedure to verify regulated nucleic acids are non-infectious may consist of introduction of the positive (+) strand RNA into permissive cells (like transfection or electroporation) to determine if the + strand RNA is capable of producing infectious virus. Consider the type of permissive cells that permit viral replication for each + strand RNA virus, the procedure of introducing + strand RNA into the cells, and appropriate positive and negative controls.

Toxicity Testing of Inactivation Procedures

Toxicity testing procedures to verify select toxins are rendered non-toxic may include, but are not limited to, functional activity assays (like the ability to cleave SNAP-25 for Botulinum neurotoxin) and in vivo exposure (like the mouse bioassay). Appropriate positive and negative controls should be included. A validated procedure, should be used to inactivate a select toxin, however the entity is not required to validate the procedure in-house.

Neutralization of Chemical Treatments Before Viability, Infectivity, or Toxicity Testing

Chemical treatments, such as fixatives and antimicrobials in inactivated samples may interfere with the viability, infectivity, or toxicity testing. Neutralization inactivates residual chemicals or antimicrobials. Therefore, viability tests should be performed once chemical or antimicrobial treatments have been subjected to neutralizing or have been shown not to interfere with the viability test.

The following experiment can be performed to determine if the chemical or antimicrobial treatment is affecting the viability, infectivity, or toxicity test:

• Split the sample in half.
• To one half, add live agent to determine if the chemical or antimicrobial activity interferes with the viability testing protocol.
• If the results demonstrate that the chemical or antimicrobial activity interfere with the viability testing protocol, perform neutralization.

If the experiment demonstrates that the residual chemicals are interfering with the viability, infectivity, or toxicity test, then neutralization should be performed. Two commonly used neutralizing media for chemical disinfectants are Letheen media and D/E Neutralizing media. Letheen media contains lecithin to neutralize quaternaries and polysorbate 80 (Tween 80) to neutralize phenolics, hexachlorophene, formalin, and, with lecithin, ethanol. D/E Neutralizing media will neutralize a broad spectrum of antiseptic and disinfectant chemicals such as aqueous formaldehyde.

The entity can use dialysis, washing of the cells, or dilution of samples to remove chemical treatments (like antibiotics or lysis buffers) that may interfere with viability testing. However, an entity must demonstrate that dialysis, washing, or dilution will neutralize residual chemical activity.

For more information on neutralization, see the Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008.